Non-heme iron (NHFe) enzymes are ubiquitous in nature and function in DNA repair, hypoxia regulation, antibiotic and natural product biosynthesis, and bioremediation, and include the anticancer drug bleomycin (BLM). On a molecular level, depending on the enzyme, the Fe II site activates dioxygen for electrophilic aromatic substitution (EAS), mono- and dioxygenation, H-atom abstraction (HAA), hydroxylation, halogenation, desaturation, and ring closure, expansion, and cleavage. They are broadly divided into 6 classes: the a- ketoglutarate (aKG) dependent enzymes, the pterin dependent enzymes, the Rieske dioxygenases, the extradiol dioxygenases, cofactor independent NHFe enzymes that act on redox inactive substrates, and BLM. These had been generally difficult to study due to their limited spectral features; in particular, they lack intene absorption features and have integer spin ground states that often cannot be studied by EPR. In comparison, heme enzymes do have intense absorption features due to the pp* transitions of the porphyrin ligand; however, these obscure the features associated directly with the Fe center and its activation of O2. This research program is directed toward developing new spectroscopic methods to elucidate the NHFe active sites, their interactions with cosubstrates, and the nature of their oxygen intermediates. New techniques are also being developed to spectroscopically access the iron in the highly covalent heme environment. Coupled to electronic structure calculations, these studies define the molecular mechanisms of the different classes of NHFe enzymes to elucidate general strategies in O2 activation. Effort is also directed toward experimentally determining how the highly delocalized porphyrin environment in heme enzymes impacts O2 activation relative to the NHFe enzymes. Ultimately, these studies will define how structurally similar NHFe sites control a wide range of reactivities and will provide fundamental insight into geometric and electronic structure contributions to determining differences in function.